Several breakthrough medical assays depend on an ability to quantify amounts of DNA in a sample. For example, copy number variation assays can be used to type cancers. For a patient identified as having lung cancer, a copy number variation assay can determine whether the patient has non-small-cell cancer by measuring the number of extra copies of epidermal growth factor receptor (EGFR) genes present is a sample from a patient. Based up the copy number variation results, a prognosis can be made quicker and suitable treatment can be started. Other medical assays, such as gene expression assays, use quantitative DNA detection to assess the progression of a disease. For example, a blood sample may be assayed for absolute amounts of DNA corresponding to biomarkers indicative of a disease, e.g., cystic fibrosis. The quantity of biomarkers from an identified panel can give information about the stage of the disease or whether treatments are improving the disease.
DNA quantification is also useful for evaluating the DNA from a sample prior to performing expensive analytical assays such as sequencing. For example, formalin-fixed, paraffin-embedded (FFPE) tissue specimens, which have been stored at room temperature for years can provide a wealth of genetic material for various molecular biology studies, such as expression profiling and sequence analysis. However, the DNA in some FFPE tissue samples degrades extensively during the storage, while the DNA in other samples is mostly intact. The amount of degradation can severely diminish the value of sequencing results. The ability to know prior to sequencing (or PCR) how much valuable DNA is present avoids wasting resources on samples without recoverable DNA. Pre-sequencing evaluation of the quantity of DNA present is also helpful in forensic science, where a blood stain, etc., may not have valuable DNA present.
Improved methods for quantifying DNA are available. In particular, real-time PCR has greatly improved the analysis of DNA from both throughput and quantitative perspectives. While traditional PCR typically relies on end-point, and sometimes semi-quantitative, analysis of amplified DNA targets via agarose gel electrophoresis, real-time PCR (or qPCR) is geared toward accurately quantifying exponential amplification as the PCR reaction progresses. Typically, qPCR reactions are monitored either using a variety of highly sequence-specific fluorescent probe technologies, or by using non-specific DNA intercalating fluorogenic dyes.
Stochastic sampling of PCR results in counting errors, especially when the starting material has little DNA, or when the sample containing the DNA targeted for counting also has large amounts of background DNA. Stochastic errors arise when random fluctuations are amplified, as is the case when a DNA sample is amplified during or before counting. In some instances, a target DNA will be missed in the first round of amplification leading to a final DNA count smaller than it should be. In other instances, a non-target DNA will be mistakenly amplified in the first round (or subsequent rounds) of amplification leading to a final DNA count higher than it should be. Thus, the resulting biased post-amplification DNA count does not represent the true condition of the sample from which it was obtained. Such errors have real consequences when the counting assay is relied upon for directing treatment for a disease.
Digital PCR (dPCR) is an alternative quantitation method in which dilute samples are divided into many separate reactions. See for example, Brown et al. (U.S. Pat. Nos. 6,143,496 and 6,391,559) and Vogelstein et al. (U.S. Pat. Nos. 6,440,706, 6,753,147, and 7,824,889), the content of each of which is incorporated by reference herein in its entirety. Typically, dPCR is conducted in a “terminal dilution” regime wherein there are at least two containers for each target molecule, however in practice there are typically more than two containers for each target molecule. At terminal dilution, the vast majority of reactions contain either one or zero target DNA molecules. The principle advantage of digital compared to qPCR is that it avoids any need to interpret the time dependence of fluorescence intensity—an analog signal—while avoiding the uncertainty of non-exponential amplification during early PCR cycles. That is, PCR amplifying a partitioned sample in the terminal dilution regime should be “all or nothing;” either a target DNA was in the partitioned sample or not. Additionally, it is more reliable to assess a “yes/no” answer with respect to a fluorescent event, as opposed to correlating a fluorescence intensity with a number of fluorescent moieties.
Nonetheless, digital PCR methods are still subject to stochastic sampling errors during sample partitioning. That is, some partitioned samples will contain more than one target DNA molecule, skewing counting methods based upon the digital readout. See, Fu et al., “Counting Individual DNA Molecules by the Stochastic Attachment of Diverse Labels,” PNAS, 108(22), 9026-9031 (2011), incorporated by reference herein in its entirety. In the terminal dilution regime, this error is negligible when there is sufficient target DNA to achieve a meaningful number of counts. When there is little target DNA in the original sample, however, the stochastic errors become meaningful, and the resultant DNA counts must be reported with much larger errors. See Fu et al.